GSM7839619: DT-resistant, rep5; Homo sapiens; RNA-Seq
Total RNA from 1x106 sensitive or resistant WM1366 cells, which were seeded 72 h beforehand in RPMI containing DMSO (sensitive cells) or BRAFi/MEKi (resistant cells), was isolated according to instructions of the RNeasy Plus Mini Kit (Qiagen). Briefly, cells were immediately lysed by addition of 350 µl buffer RLT Plus including 1% β-mercaptoethanol and resuspended by repeatedly pipetting up and down. Following the procedure of the manufacturer, RNA was isolated and finally eluted with 50 µl RNase-free water For library preparation, total RNA with an integrity number of ≥ 9 was used. mRNA was isolated from 500 ng total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacturer's instructions (NEB). Final elution was done in 15ul 2x first strand cDNA synthesis buffer (NEBnext, NEB). After chemical fragmentation by incubating for 15 min at 94°C the sample was directly subjected to the workflow for strand specific RNA-Seq library preparation (Ultra Directional RNA Library Prep, NEB). For ligation custom adaptors were used (Adaptor-Oligo 1: 5'-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT-3', Adaptor-Oligo 2: 5'-P-GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT CAC-3'). After ligaton adapters were depleted by an XP bead purification (Beckman Coulter) adding bead in a ratio of 1:1. Indexing was done during the following PCR enrichment (12 cycles) using custom amplification primers carrying the index sequence indicated with 'NNNNNN'. (Primer1: Oligo Seq AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T, Primer2: GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T, Primer3: CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN GTG ACT GGA GTT). After two more XP beads purifications (1:1) libraries were quantified using Qubit dsDNA HS Assay Kit (Invitrogen, Waltham, USA). For Illumina flowcell production, samples were equimolarly pooled sequenced at 40 Mio fragments 100bp paired-end on a NovaSeq 6000 device using a NovaSeq v1.5 S4 flowcell.